Pinkee Pandey et al. / Journal of Pharmacy Research 2011,4(6),1735-1737 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Antioxidant property, total Phenolic content and inhibition of a-amylase activity of graveolens L. leaves Pinkee Pandey*, Archana Mehta, Subhadip Hajra, Jinu John and Pradeep Mehta School of Biological and Chemical Sciences, Dept. of Botany Dr. H. S. Gour Central University, Sagar 470003, Madhya Pradesh, India Received on: 11-02-2011; Revised on: 16-03-2011; Accepted on:21-04-2011 ABSTRACT In the present study we evaluate the antioxidant and antidiabetic activity of ethanolic extract of leaves using various in-vitro models. Phenolic compounds of origin are of great interest due to their antioxidant property and serves as essential part of human diet. The alcoholic leaves extract of Ruta graveolens showed DPPH radical scavenging activity 08.48, 10.45, 11.15,13.01 and 19.37% at respective concentrations of 10, 50, 100, 250 and 400 µg/ml and the results were compared with that of standard drug Butylated Hydroxyl Anisole (BHA). Also the extract showed relatively better hydroxyl radical scavenging activity of 42.10, 47.90, 58.71, 61.61and 72.67% at respective concentrations. The total phenolic content of leaves extract was found to be 13µg/mg Catechol equivalent when assayed by Folin-Ciocalteau method. The extract significantly inhibited nitric oxide, superoxide anions and showed ferric reducing power. The extract exhibited a- amylase inhibition in a concentration dependent manner. At concentrations of 2, 20 and 200 µg/ml extract showed 70.78, 72.23 and 72.53% inhibition. The results obtained in this study clearly indicate that R. graveolens has a significant potential to use as a natural antioxidant as well as antidiabetic agent.

Key words: Alcoholic extract, Ruta graveolens, antioxidant, antidiabetic, phenolic INTRODUCTION - 2.2. Plant material Reactive oxygen species (ROS) such as anion radicals (O2 ), hydroxyl radical (OH) and non-free 1 The leaves of R. graveolens were collected in the month of September from Sagar radical species such as H2O2 and singlet oxygen ( O2), are various forms of activated oxygen capable of damaging DNA, , carbohydrates and lipids. ROS are involved in various District, Madhya Pradesh, India. Further taxonomic identification was conducted at physiological and pathological events such as inflammation, aging, mutagenicity and carcino- Department of Botany. Dr. H.S. Gour Central University, Sagar, MP, India. genicity [1]. Free radicals activities are controlled by a system of endogenous enzymatic and non- enzymatic antioxidants which eliminate pro-oxidants and scavenge free radicals [2]. 2.3. Preparation of the extract Antioxidants are compounds that can delay or inhibit the oxidation of lipids or other molecules The air-dried leaves of R. graveolens (20 g) were powdered and soaked in 300 ml of 75% by inhibiting the initiation or propagation of oxidative chain reactions. The anti-oxidative of and kept in a shaker for 72 h. The crude extract was collected by filtration and effect of plant are mainly due to phenolic components, such as flavonoids, phenolic evaporated under reduced pressure to give a blackish green amorphous mass. Extract was acids and phenolic diterpenes. Antioxidant Agents of natural origin have attracted special dissolved in sterile distilled water to get desired concentrations for the studies. interest because they can protect human body from free radicals. Numerous medicinal and their formulations are used for liver disorders in ethnomedical practices as well as in 2.4. Phytochemical analysis traditional systems of medicine in India. Phytochemicals screening were performed to detect various compounds such as tannins, flavonoids, alkaloids and steroids etc.,[3]. mellitus is a chronic metabolic disorder characterized by abnormalities in carbohy- drates, lipid, and lipoprotein metabolism, cause many complications, such as hyperlipidemia, 2.5. Determination of total phenolic content hyperinsulinemia, hypertension, and atherosclerosis. Nutritional factors including antioxi- Total soluble phenolic content in the extract was determined with Folin–Ciocalteau dants have great influence in the management of diabetes mellitus and its complications. An reagent [4]. Aliquot of each sample were pipetted out in series of test tubes and volume was imbalance between oxidative stress and anti- oxidative defense mechanisms in diabetics can made up to 3 ml with distilled water. 0.5ml of Folin-Ciocalteau reagent was added to each result in cell and tissue damage and accelerate diabetic complications. Oxidative stress may tube, mixed thoroughly and incubated for 3 min. at room temperature. 2ml Sodium have significant effect in the transport (GLUT) or at insulin receptor. Scavengers carbonate (20%) solution was added and the mixer was incubated for 1 min. in boiling of oxidative stress may have an effect in reducing the increased serum glucose level in diabetes water bath. Absorbance was measured at 650 nm against a reagent blank. Standard curve and may alleviate the diabetes as well as reduce its secondary complications. was prepared using catechol as standard phenolic compound. From the standard curve, concentration of phenols in the test samples was determined and expressed as µg of Ruta graveolens commonly known as rue, is a dicot , belongs to family and catechol equivalent. native to Mediterranean region but widely distributed all over the tropical regions. The leaves are bipinnate or tripinnate with a feathery appearance and green to strongly glaucous blue-green 2.6. Determination of DPPH (1-1-diphenyl 2-picryl hydrazyl) radical scavenging in colour. This plant is used by the Jordanianpopulations, systemically for its antispasmodic activity and analgesic effects and externally for its antirheumatic activity. The , petroleum The free radical-scavenging activity of the extract was measured in terms of hydrogen ether, ethyl acetate and water-methanol extracts of R. graveolens were found to possess donating or radical-scavenging ability [5]. Different concentrations (10, 50,100, 250 and 400 µg/ml of extract and reference drug BHA were mixed with 5 ml of methanolic solution antimicrobial and cytotoxic activities. Ruta in combination with Ca3 (PO4)2 is found to be effective in treatment of brain cancers, particularly glioma. Leaf extracts also reported to possess of DPPH (0.1 mM). The test solutions were allowed to stand at room temperature for 20 strong anti-inflammatory activity [11]. min. The absorbance of the samples was measured at 517 nm. Reagent solution without test samples was used as control. Radical scavenging activity was calculated by using the However, no significant reports are available about the antioxidant and antidiabetic activity formula: of Ruta graveolens leaves; therefore, present investigation was made to examine the total ( Control OD - Sample OD) phenolic content, antioxidant and antidiabetic activities of ethanolic leaves extract of Ruta % radical scavenging activity = x100 graveolens through various in vitro models. Control OD 2.7. Determination of hydroxyl radical scavenging activity 2. MATERIALS AND METHODS Hydroxyl radical scavenging activity was measured according to [5]. Various concentrations 2.1. Chemicals 10, 50, 100 and 250 and 400 µg/ml of samples were taken in different test tubes and The major Chemicals used for the study includes Nitro blue tetrazolium (NBT), 2,2- volume was made up to 250µl with 0.1M phosphate buffer. 1 ml of iron-EDTA solution diphenyl-1-picrylhydrazyl (DPPH) (Sigma, Germany), Gallic acid (Loba Chemie, Mumbai), (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 ml of EDTA (0.018%), and 1 Sodium carbonate, Sodium nitroprusside (HIMEDIA) and Trichloro acetic acid (S.d-fine ml of Dimethyl sulphoxide (0.85% v/v in 0.1 M phosphate buffer, pH 7.4) were added to chemicals, Mumbai). All other reagents used were of analytical grade. these tubes, and the reaction was initiated by adding 0.5 ml of 0.22% ascorbic acid. These reaction mixtures were incubated at room temperature for 15 min. The reaction was terminated by the addition of 1 ml ice-cold TCA (17.5% w/v). 3 ml of Nash reagent (150 g of ammonium acetate, 3 ml of glacial acetic acid and 2 ml of acetyl acetone were mixed and made up to 1 lit. with distilled water) was added to each tubes and left at room *Corresponding author. temperature for 15 min. The intensity of the yellow color formed was measured Pinkee Pandey spectrophotometrically at 412 nm against reagent blank. The percentage of hydroxyl School of Biological and Chemical Sciences radical scavenging activity was calculated by the formula: Dr. Hari Singh Gour Central University Sagar (M.P), India 470 003

Journal of Pharmacy Research Vol.4.Issue 6. June 2011 1735-1737 Pinkee Pandey et al. / Journal of Pharmacy Research 2011,4(6),1735-1737 2.8. Determination of nitric oxide radical scavenging activity 3.4. Hydroxyl radical scavenging Various concentrations 10, 50, 100 and 250, 500 and 750µg of extracts were taken in The extract displayed potential hydroxyl radical-scavenging activity (Fig. 2). The % of different test tubes and made up to 3ml with 0.1M phosphate buffer (pH 7.2). 1 ml Sodium inhibition of extracts at concentrations of 10, 50, 100, 250 and 400µg/ml was 42.10, 47.90, Nitroprusside (5mM prepared in buffered saline pH7.2) was added to each tube. The 58.71,61.61 and 72.67% respectively. All results showed antioxidant activity in dose dependent reaction mixture was incubated for 30 min at room temparature. After 30 min, 1.5 ml of manner. IC50 value for extract and BHA was found to be 160.09µg/ml 325.25µg/ml respectively. above solution was mixed with 1.5 ml of Griess reagent (1% Sulphanilamide, 2% Ethanolic extract of rebaudiana leaves also showed similar hydroxyl radical scavenging phosphoric acid and 0.1% N-1Naphthylethylenediamine dihydrochloride). Control without activity in a concentration dependent manner [12]. Hydroxyl radicals are produced from the the test compound, but with an equivalent amount of methanol was maintained. The decomposition of hydro-peroxides (ROOH) by the reaction of excited atomic oxygen with absorbance of the samples was measured at 546 nm [6]. Nitric oxide radical scavenging water. Hydroxyl radical scavenging capacity of an extract is directly related to its antioxidant activity was calculated using the formula: activity [13].

2.9. Determination of ferric reducing power Ferric reducing scavenging activity was determined by [7]. Various concentrations of sample 100, 250, 400 and 500µg were mixed with 2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50ºC for 20 min; then 2.5 ml of 10% (w/v) trichloroacetic acid was added. 5 ml of above

solution was mixed with 5 ml of distilled water and 1 ml of 0.1% of ferric chloride. The % Inhibition absorbance was measured spectrophotometrically at 700 nm. BHA was used as standard antioxidant.

2.10. Amylase inhibitory activity Amylase inhibitory activity was perfomed by [8]. Leaves extract were dissolved in sterile distilled water and prepared different concentration (2, 20 and 200 µl). Reaction mixture containing 100µl of extract, 200µl of the porcine pancreatic a-amylase, 100µl of 2 mM phosphate were mixed and incubated at 37ºC for 10 min followed by the addition of 100 Concentration in mg/ml µl of 1% starch solution was added. After incubation for 5 min, 1 ml of DNS solution was Fig.2. Hydroxyl radical scavenging activity of the ethanolic leaves extract of Ruta graveolens added. Reagent solution without test samples was used as control. The tubes were then 3.5. Nitric oxide radical scavenging incubated in boiling water bath for 10 min, cooled and the absorbance was measured at The extract showed concentration dependent nitric oxide radical scavenging activity of 13.05, 540 nm against blank. Concentration of maltose liberated was determined by using 15.57, 20.36, 28.02, 43.01, 48.45 and 63.25% inhibition at a respective concentration of 10, standard maltose curve. Enzyme activity was calculated by following formula and expressed 50, 100, 250, 400, 500 and 750 µg/ml (Fig. 3). IC50 values for extracts and BHA was found as µmoles/min/ml. to be 540.41µg/ml and 638.01µg/ml respectively. Nitric oxide was generated from sodium nitroprusside in aqueous solution at physiological pH and interacts with oxygen to produce nitrite ions that can be estimated by Greiss reagent [14,15,16]. Nitric oxide (NO) is a potent pleiotropic mediator of physiological process such as smooth muscle relaxant, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity. It is a 3. RESULTS AND DISCUSSION diffusible free radical which plays many roles as an effectors molecule in diverse biological systems. Nitric oxide and superoxide radicals are involved in host defense; over production of 3.1. Preliminary phytochemical analysis these two radicals contribute to the pathogenesis of some inflammatory diseases [17]. Nitric Screening of ethanolic extract of Ruta graveolens leaves extract showed presence of flavonoids, oxide inhibitors have been shown to have beneficial effects on some aspect of inflammation and tannins, alkaloids and steroids. tissue damage seen in inflammatory diseases. The result indicated that the extract might contain compounds able to inhibit nitric oxide and offers scientific evidence for the use of the 3.2. Total phenolic content R. graveolens leaves in the indigenous system in inflammatory condition. The total amount of phenolic content in Ruta graveolens leaves was found to be 13µg/ml of Catechol equivalent. Phenolics are the most wide spread secondary metabolites in plant kingdom. These diverse groups of compounds have received much attention as potential natural antioxidant. The antioxidant activity of the plant extract is mainly due to presence of phenolic compounds. Phenols are very important plant constituents because of their scavenging ability due their redox properties, hydrogen donors and singlet oxygen quenchers [9]. The interests of phenolics are increasing in the industry because they retard oxidative degradation of lipids and improve the quality and nutritional value of food [10].

3.3. Inhibition of DPPH radical The extract showed concentrations depended DPPH radical scavenging activity of 08.48, 10.45, 11.15 and 13.01, 19.37% of inhibition at a respective concentration of 10, 50, 100, 250 and 400 µg/ml) (Fig.1). The 1, 1-diphenyl-2-picryl-hydrazyl (DPPH), assay used for hydrogen % Inhibition donating capacity is commonly employed for screening plant extract [11]. The DPPH radical contains an odd electron, which is responsible for the absorbance at 517 nm. When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized, which can be quantitatively measured from the changes in absorbance. The DPPH free radical scavenging of antioxidants is due to their hydrogen donating ability; plants with higher donating capacity have shown higher DPPH free radical scavenging activity. Concentration in mg/ml

Fig.3. Nitric oxide radical scavenging activity of the ethanolic leaves extract of Ruta graveolens

3.6. Ferric oxide radical scavenging The various concentrations of leaves extracts (100–500 µg/ml) showed 0.04, 0.17, 0.28 and 0.54% inhibition respectively. A 500 µg/ml of extract and BHA exhibited 0.54% and 1.59% inhibition respectively (Fig. 4). Presence of hydrophilic poly phenolic compounds in extract cause the greater reducing power. % Inhibition 3.7.Amylase inhibitory activity The ethanolic extract showed amylase inhibitory activity of 70.78, 72.23 and 72.53% (µg/ml/ min) at respective concentration of 2, 20 and 200 µg/ml (Table: 1). Amylase catalyses the hydrolysis of a -1, 4-glucosidic linkage of starch, glycogen and various oligosaccharides and glucosidase further breaks down the disaccharides into simpler sugars. a- amylase inhibitory activity in the digestive tract of humans is considered to be effective in control of diabetes by Concentration in mg/ml diminishing the absorption of glucose [18]. Therefore, the result shows that R. graveolens Fig. 1. DPPH radical scavenging activity of the ethanolic leaves extract of Ruta graveolens Journal of Pharmacy Research Vol.4.Issue 6. June 2011 1735-1737 Pinkee Pandey et al. / Journal of Pharmacy Research 2011,4(6),1735-1737 Table 1: Inhibition of amylase in presence of different concentrations of ethanolic Further studies regarding the in vivo studies, isolation and characterization of the active extract of Ruta graveolens leaves principles responsible for antioxidant and antidiabetic activity is currently under progress.

Concentration (µg/ml) % Inhibition ACKNOWLEDGEMENTS Authors are grateful to Head Department of Botany, Dr. H.S. Gour University, Sagar, 2 70.78 M.P., India, for providing laboratory facilities and University Grant Commission (UGC), 20 72.23 New Delhi, India, for providing financial assistance. 200 72.53 REFERENCE [1]. Arouma OI. Characterization of drugs as antioxidant prophylactics. Free Radic. Biol. Med 8, (1996), 95-9. [2]. Chait A, Brunzell JD. Diabetes, lipids, and atherosclerosis. In: LeRoith, D., Taylor, S.I., Olefsky, J.M., (Eds.), Diabetes Mellitus. Lippincott-Raven Publishers, Philadelphia, (1996) 467–469. [3]. Evans WC, Trease and Evan’s Pharmacognosy. Thirteenth Edition. English Language Book Society/ Baillere Tindall. London, (1999), 327- 536. [4]. Malick CP, Singh M.B. In: Plant Enzymology and Histoenzymology. Kalyani Publishers New Delhi, (1980), 286. [5]. Singh RP, Murthy KNC. Jayaprakasha GK. Studies on the antioxidant activity of (Punica granatum) peel and extracts using in vitro models. J. Agric. Food Chem 50, (2002), Absorbance 81-86. [6]. Kumar S, Kumar D, Manjusha, Saroha K, Singh N, Vashishta B. Antioxidant and free radical scavenging potential of Citrullus colocynthis (L.) Schrad. methanolic fruit extract. Acta Pharmaceutica 58, (2008), 215–220. [7] Barreira JCM, Ferreira ICFR, Oliveira MBPP, Pereira JA, Antioxidant activity and bioactive compounds of ten Portuguese regional and commercial almond cultivars. Food Chem. Toxicol 46, (2008), 2230-2235. [8]. Sadasivam S, Manickam A. Biochemical Methods, II edition, New Age International (P) Ltd, publishers, New Delhi, (2006), 124-126. [9]. Hatano T, Edamatsu R, Mori A. Effects of interaction of tannins with coexisting substances. Chem. Concentration in mg/ml Pharm. Bull 37, (1989), 2016–2021. Fig. 4. Ferric oxide radical scavenging activity of the ethanolic leaves extract of Ruta graveolens [10] Aneta W, Jan O, Renata C. Antioxidant activity and phenolic compounds in 32 selected . Food Chem 105, (2007), 940–949. [11]. Soares JR, Dinis TCP, Cunha AP, Almeida LM. Antioxidant activities of some extracts of Thymus leaves extract can act as an effective and nontoxic inhibitor of amylase and glucosidase which zygi. Free Radic. Res 26, (1997), 469–478. indirectly posses its antidiabetic potential. [12]. Shukla S, Mehta A, Bajpai VK, Shukla S. In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Bert. Food Chem. Toxicol 47, (2009), 2338–2343. 4. CONCLUSION [13]. Babu BH, Shylesh BS, Padikkala J. Antioxidant and hepatoprotective effect of Alanthus icicifocus. Fitoterapia 72, (2001), 272–277. The ethanolic extract of R. graveolens leaves showed strong antioxidant activity by inhibiting [14]. Green LC, Wagner DA, Glogowski J. Analysis of nitrate, nitrite and nitrate in biological fluids. Anal. DPPH, hydroxyl radical, nitric oxide and ferric oxide scavenging activities when compared Biochem 126, (1982),131–138. with standard drug BHA. R. graveolens leaf extract possesses free radical scavenging activity [15]. Marcocci L, Maguire JJ, Droy-Lefaix MT, Packer L. The nitric oxide-scavenging properties of which could exert a beneficial action against liver damage induced by different exogenous and Ginkgo biloba extract EGb 761. Biochem. Biophys Res. Commun 15, (1994), 748–755. [16]. Marcocci L, Packer L, Droy-Lefaix MT. Antioxidant actions of Ginkgo biloba extract EGB 761. endogenous sources. The present study suggests that phenolic compounds of the R. graveolens Methods Enzymol 234, (1994), 462–475. leaves provide a good source of antioxidants that could offer potential protective effects against [17]. Guo X, Wang WP, Ko J K, Cho CH, Involvement of neutrophils and free radicals in the potentiating lipid oxidation. The plant extract also showed significant antidiabetic activity in a concentration effects of passive cigarette smoking on experimental inflammatory bowel disease in rats. dependent manner. These results remain important as the first step in screening antioxidant and Gasteroenterology 117, (1999), 884-892. [18]. Hara Y, Honda M. The inhibition of a-amylase by tea polypphenols. Agric Biol. Chem 54, (1990), antidiabetic activity of R. graveolens leaves. Thus, it can be concluded that ethanolic extract 1939–1945. of R. graveolens leaves can be used as an accessible source of natural antioxidants and antidiabetic agent with consequent health benefits. Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 6. June 2011 1735-1737